Indirect Proximity Ligation Assay (PLA) - Brightfield
Ayse Ulusoy, Rita Pinto-Costa, Angela Rollar, Donato Di Monte
Abstract
Indirect Proximity Ligation Assay (PLA) is a powerful molecular technique used to detect and visualize protein-protein interactions, protein modifications, and protein complex formations within cells or tissues. This method is based on the principles of proximity-dependent ligation and utilizes specific antibodies to detect the nitration of proteins on free-floating brain sections. Here we describe the PLA protocol that we routinely use in our laboratory to detect nitrated alpha-synuclein and nitration of mitochondrial enzymes such as SOD2 and the mitochondrial complex 1 subunit NDUFB8.
Steps
Day 1
Pick 35um cut brain sections and transfer them to1.5mL Eppendorf tubes
Note: all incubation and wash steps are performed by shaking Eppendorf tubes at 250rpm (e.g., thermomixer)
Wash 2x0h 5m 0s with Tris-HCl
Peroxidase quenching with BloxAll solution
use300µL and incubate for 30min
Wash 3x0h 5m 0s with wash buffer A (see materials)
Antigen-retrieval with citrate buffer (+tween, pH = 6) at 95°C for 0h 4m 0s
Note: Heat the solution to 95°C before adding on the samples
Wash 3x0h 5m 0s with wash buffer A
Blocking: Incubate in Duolink Blocking Solution (300µL)at 37°C for 1h 0m 0s
Primary Antibody Incubation : Dilute antibodies in Duolink Antibody Diluent (200µLµl/tube) and incubate 0h 5m 0s room temperature.
for nitrated alpha-synuclein: mouse anti-3-NT and rabbit anti-h-alpha-synuclein
for nitrated SOD2: mouse anti-3-NT and rabbit anti-SOD2
for nitrated NDUFB8: mouse anti-3-NT and rabbit anti-NDUFB8
see materials for dilutions and catalog numbers
Day 2
Wash 3x0h 10m 0swith wash buffer A
PLA probe incubation :
Prepare anti-mouse MINUS and anti-rabbit PLUS probes following manufacturer's instructions, add solution on sections and incubate at 37°C for1h 30m 0s
Wash 3x0h 10m 0s with wash buffer A
Ligation:
Dilute the Duolink Ligation Buffer 1:5 in high-purity water
Add the ligase (diluted 1:40) just before incubation (keep cold on freezer block!)
add the ligation solution on sections and incubate at 37°C for 1h 15m 0s
Wash 3x0h 5m 0s with wash buffer A
Amplification :
Dilute the Duolink Amplification Buffer 1:5 in high-purity water
Add the polymerase (diluted 1:80) just before incubation (keep cold on freezer block!)
Add the amplification solution to the sections and incubate at 37°C for 2h 15m 0s
Detection:
Dilute the Duolink Detection Brightfield Stock 1:5 in high-purity water
add solution to the sections and incubate at RT for1h 10m 0s
Wash 2x0h 5m 0s with wash buffer A
Wash 1x0h 5m 0s with Tris-buffered saline (no detergent)
Developing:
Dilute the Duolink Brightfield Substrate solutions in high-purity water:
§ Substrate A (1:70)
§ Substrate B (1:100)
§ Substrate C (1:100)
§ Substrate D (1:50)
Incubate sections at RT for 1-5min (depending on desired intensity outcome)
Wash 3x0h 5m 0s with wash Tris-buffered saline
Mount the samples on slides and let them air-dry
Nuclear staining (optional): Incubate for 0h 1m 0s
Wash the staining off with running tap water for 0h 10m 0s
Dehydrate of samples in 1min each: 70% ethanol, 95% ethanol, 2x 100% ethanol 2x xylene
Coverslip samples with Histomount mounting medium
